Buffered wash composition, insolubilizing composition, test kits and method of use

ABSTRACT

A buffered aqueous composition is useful simultaneously as a wash solution and a dye-providing composition in specific binding assays involving enzyme-labeled specific binding reagents. The wash composition includes a dye-providing composition, a buffer and an organic solvent having a certain molecular weight and water-solubility. Another useful composition includes a particulate substrate having avidin attached thereto, and a peroxidase reducing agent. Either composition can be provided in a diagnostic test kit, and can be used to detect a specific binding ligand in assays.

FIELD OF THE INVENTION

This invention relates to a composition which is useful as a washsolution and dye-providing composition simultaneously in specificbinding assays. Further, an immobilizing composition is provided. Thisinvention also relates to a diagnostic test kit containing eithercomposition and to methods for determining specific binding ligands.

BACKGROUND OF THE INVENTION

There is a continuous need in medical practice, research and diagnosticprocedures for rapid and accurate determinations of biologicalsubstances which are present in biological fluids at low concentrations.For example, the presence of hormones, drugs, narcotics, steroids,polypeptides, prostaglandins, proteins, antibodies or infectiousorganisms in blood, saliva, urine or other biological fluids must bedetermined in an accurate and rapid manner for suitable diagnosis ortreatment.

To provide such determinations, various methods have been devised forisolating and identifying biological substances employing specificbinding reactions between the substance to be determined (identifiedherein as a "ligand"), and receptor molecules specifically reactive withthe ligand. Radioisotopes, fluorogens, chromogens, detectable beads andenzymes have been used to detect the resulting complex between ligandand receptor. One common example of specific binding reactions is animmunoassay in which an antigenic substance and specific antibodythereto react to form an immunological complex.

In recent years, the use of enzyme labels has received increasedattention for specific binding assays because of various disadvantagesassociated with radioactive and fluorescent labels. Assays using enzymelabels include what are known in the art as competitive enzymeimmunoassays (EIA) and both direct and indirect enzyme linkedimmunosorbent assays (ELISA). Another type of useful assay is known asan immunometric or "sandwich" assay, as exemplified in U.S. Pat. No.4,486,530 (issued Dec. 4, 1984 to David et al). In all of these assays,either a receptor for the ligand, or a known quantity of ligand analogis labeled with a enzyme so that ligand-receptor complexes can bedistinguished from unlabeled materials. Generally, the complexes areseparated from uncomplexed materials using some type of immobilizingtechnique with or without washing or filtration.

Peroxidase is one enzyme which has been used to advantage as a label inanalytical methods. Peroxidase acts on hydrogen peroxide as a substrateand can oxidize various chromogens or dye-providing materials to providea detectable species at a rate proportional to the amount of peroxidasepresent. Various dye-providing materials are known in the art, includingbenzidine and its derivatives, and various leuco dyes.

U.S. Ser. No. 136,166 (filed Dec. 18, 1987 by McClune and Bishop, nowU.S. Pat. No. 5,024,935 issued Jun. 18, 1991) describes an improveddye-providing composition which is stabilized with certain polymers. Thecomposition includes dye-providing leuco dyes dissolved in methanolwhich can be converted to detectable dyes in the presence of peroxidaseand hydrogen peroxide. While the described assay has found significantusefulness in the art, particularly for pregnancy tests, separate washand dye-providing solutions and steps are required for effectiveresults. It would be desirable to eliminate steps and solutions in orderto make the test more reliable and simple for the user.

SUMMARY OF THE INVENTION

The useful assay described in U.S. Pat. No. 5,024,935 (noted above) isfurther improved using a buffered aqueous wash composition comprising:

a. a composition capable of providing a dye in response to an enzymewhich is the label on a specific binding reagent,

b. a buffer, and

c. a water-soluble organic solvent which has a molecular weight betweenabout 40 and about 100 and is present in an amount of from about 2.5 toabout 25 volume %.

Also provided by this invention is a composition for insolubilizing abiotinylated specific binding reagent, the composition comprising aparticulate substrate having avidin attached thereto and a reducingagent for peroxidase. This immobilizing composition can be included in adiagnostic test kit, with or without the wash composition describedherein.

This invention also provides a diagnostic test kit comprising:

a. an enzyme-labeled receptor for a specific binding ligand, and

b. a buffered aqueous wash composition comprising:

a. a composition capable of providing a dye in response to the enzyme,

b. a buffer, and

c. a water-soluble organic solvent which has a molecular weight betweenabout 40 and about 100 and is present in an amount of from about 2.5 toabout 25 volume %.

Further, a method for the determination of a specific binding ligandcomprises the steps of:

A. contacting a specimen suspected of containing a predeterminedspecific binding ligand with an enzyme-labeled receptor for the ligandto form a detectable complex of ligand and receptor,

B. washing the detectable complex with a buffered aqueous washcomposition comprising:

a. a composition capable of providing a dye in response to the enzyme,

b. a buffer, and

c. a water-soluble organic solvent which has a molecular weight betweenabout 40 and about 100 and is present in an amount of from about 2.5 toabout 25 volume %, and

C. detecting the resulting dye as an indication of the presence of thespecific binding ligand in the specimen.

The present invention provides an improved composition which provides adye in the presence of an enzyme-labeled specific binding reagent, andwhich is also useful as a wash solution in a specific binding assay.This composition can be used to advantage in assays where any enzyme isused as the label, but particularly when peroxidase is used as thelabel. Because the composition is useful as a wash solution as well as adye-providing solution, separate solutions and steps are eliminated.Thus, the assay is simplified, assay time is reduced and the likelihoodfor error is reduced. The composition of this invention can be readilypackaged in a diagnostic test kit which has less components than knownkits.

The advantages of this invention are provided in a specially formulatedwash composition including a dye-providing composition responsive to anenzyme label, buffer and water-soluble organic solvent which has amolecular weight from about 40 to about 100, and which is present in thecomposition at a volume percent of about 2.5 to about 25.

When peroxidase is used as the preferred enzyme label, it is necessaryto include a reducing agent which will inhibit the peroxidativeformation of dye while removing excess peroxidase during the wash step.Advantageously, in some embodiments in which a biotinylated receptor isused, an insolubilizing composition includes a particulate substrate towhich avidin is attached, and a peroxidase reducing agent.

Detailed Description of the Invention

The wash composition of the present invention is useful for providing adye in the presence of a an enzyme used as a label on a specific bindingreagent, such as an antigen, antibody, hapten or drugs. Representativeenzymes include peroxidase, glucose oxidase, alkaline phosphatase,glucosidase, urease, β-glucosidase, β-galactosidase and others known toone skilled in the art. Techniques for attaching such enzymes tospecific binding reagents are well known. This composition isparticularly useful with conjugates of peroxidase and the specificbinding reagent, such as a peroxidase-labeled antibody. Preferably, thewash composition of this invention is used in assays which involvespecific binding reactions, such as immunoassays, as described in moredetail below.

The wash composition of this invention includes a dye-providingcomposition which, in turn, includes one or more reagents which providea dye upon interaction of the enzyme with the appropriate substrates. Insome instances, the dye-providing composition is a single reactant whichboth provides a dye and is the needed substrate for the enzyme. In otherembodiments, two or more reagents are needed for enzymatic activity anddye formation.

Depending upon the enzyme used, the dye-providing composition will varyin components. A worker or ordinary skill in the art would know whatreagents are needed for a given enzyme, and useful amounts. for example,if glucose oxidase is the label, the dye-providing composition caninclude an aniline and oxidizable compound to provide a dye. Foralkaline phosphatase, a suitable reagent includes a phosphate substratewhich will directly or indirectly provide a dye.

In the preferred embodiment where the enzyme is peroxidase, any suitableperoxidase-reactive substrate and dye-former can be used, includingtetrabenzidine and its derivatives which are well known in the art.Preferably, the composition includes one or more leuco dyes which arecapable of providing a dye in the presence of hydrogen peroxide and aperoxidative substance (that is, peroxidase or a substance that actslike peroxidase). The resulting dye is generally detectable in thevisible region of the electromagnetic spectrum (generally from about 400to about 700 nm). Preferably, the dye is detected at from about 500 toabout 650 nm.

Imidazole leuco dyes useful herein are either diarylimidazole ortriarylimidazole leuco dyes. Many useful compounds are known in the art,including those described in U.S. Pat. No. 4,089,747 (issued May 16,1978 to Bruschi) and references noted therein, EP-A-0 122 641 (publishedOct. 24, 1984) and Japanese Patent Publication 58(1983)-045,557.

The triarylimidazoles having the following general formula areparticularly useful: ##STR1## wherein R¹, R² and R³ are each an organicgroup such that at least one of them is an ortho- or para-hydroxysubstituted aryl group of up to 18 carbon atoms, the other two groupsbeing aryl groups chosen such that the imidazole oxidation potential isbetween about -70 and +110 mV as measured by cyclic voltammetry againsta standard calomel electrode using a carbon based electrode. Oxidationpotential measurements can be made according to conventionalelectrochemical techniques (see, for example, Sawyer et al, ExperimentalElectrochemistry for Chemists, John Wiley & Sons, New York, 1974).

As used herein, the term "aryl" is meant to include aromatic hydrocarbongroups, such as phenyl, naphthyl or anthryl, tolyl, xylyl and othersubstituted aromatic groups. The number of carbon atoms refers to thetotal number of nuclear carbon atoms as well as those in substituents.At least one of the R¹, R² and R³ groups has an ortho or para electrondonating substituent such as an alkoxy (--OR) wherein R is alkyl of 1 to8 carbon atoms (for example, methyl, ethyl, isopropyl, t-butyl, hexyl,chloromethyl or methoxymethyl), or a dialkylamino wherein alkyl is asjust defined. The R¹, R² and R³ groups can have one or more othersubstituents which are electronically compatible with the imidazolenucleus to provide a suitable dye upon oxidation. Further details ofpreferred triarylimidazole compounds and methods of preparing them arefound in U.S. Pat. No. 4,089,747 noted above.

Particularly useful triarylimidazole leuco dyes are selected from thegroup consisting of:

2-(4-hydroxy-3,5-dimethoxyphenyl)-4,5-bis(4-methoxyphenyl)imidazole,

2-(3,5-dibromo-4-hydroxyphenyl)-4,5-diphenylimidazole,

2-(3-bromo-5-methoxy-4-hydroxyphenyl)-4,5-bis(4-methoxyphenyl)imidazole,

4,5-bis(4-dimethylaminophenyl)-2-(4-hydroxyphenyl)imidazole,

4,5-bis(4-dimethylaminophenyl)-2-(4-hydroxy-3-methoxyphenyl)imidazole,

2-(4-hydroxyphenyl)-4,5-bis(4-methoxyphenyl)-imidazole, and

4,5-bis(4-dimethylaminophenyl)-2-(4-hydroxy)-3,5-dimethoxyphenylimidazole.

The amount of leuco dye in the preferred wash composition can be variedwidely. Generally, it is present in an amount of from about 10⁻⁶ toabout 10⁻³ and preferably from about 10⁻⁵ to about 10⁻⁴ molar.

The wash composition of this invention is generally buffered to a pH offrom about 6 to about 9, depending upon the assay it is being used for.One or more buffers can be used, and suitable buffers are known in theart including, but not limited to phosphates, borates,3-(N-morpholine)propanesulfonic acid, tris(hydroxymethyl)aminomethane,N-tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid,N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic) and others readilyapparent to one skilled in the art. The amount of buffer can be readilydetermined to provide the desired pH and buffering capacity. Generally,it is present in an amount of at least about 10 mmolar.

Moreover, the wash composition also includes one or more water-solubleorganic solvents having a molecular weight of from about 40 to about100. Such solvents are polar and have a solubility in water of at least10% (by volume) at room temperature. Some of the solvents arewater-miscible, while others have more limited water-solubility.

Useful solvents must not interfere with the assay in any way, or bedetrimental to washing uncomplexed materials or dye formation. A modestexperiment may be performed to determine if a particular solvent isuseful in the practice of the invention. Particularly useful solventsare the lower alcohols such as ethanol, n-propanol, isopropanol,n-butanol, sec-butanol, tert-butanol and others known to one skilled inthe art. Other useful solvents include acetonitrile, ketones such asacetone and methyl ethyl ketone, and ethers such as tetrahydrofuran and1,4-dioxane. Others would be readily apparent to one skilled in the art.The preferred solvents are the lower alcohols with sec-butanol beingmost preferred.

The water-miscible solvent is generally present in the composition in anamount of from about 2.5 to about 25, and preferably from about 5 toabout 15, volume percent.

In the preferred embodiments wherein the wash composition includes aleuco dye, it also preferably includes one or more water-soluble orwater-dispersible polymers, such as vinyl pyrrolidone polymers,acrylamide polymers, acrylic and methacrylic acid polymers, polyethyleneglycols and polyamines. These polymers can be either homo- orcopolymers. Representative examples of useful polymers include, but arenot limited to: poly(acrylic acid), poly(methacrylic acid), poly(acrylicacid-co-methyl acrylate) (90:10 weight ratio), poly(acrylamide),poly(acrylamide-co-acrylic acid) (50:50 weight ratio), polyamines suchas those described in U.S. Pat. Nos. 3,702,249 and 4,689,359.Particularly useful polymers are vinyl pyrrolidone polymers, that is ahomo- or copolymer prepared from vinylpyrrolidone such aspoly(vinylpyrrolidone), poly(vinylpyrrolidone-co-acrylic acid) andpoly(vinylpyrrolidone-co-acrylamide). Further details regarding thesepolymers are provided in U.S. Pat. No. 5,024,935 (noted above),incorporated herein by reference.

Other optional components of the wash composition include electrontransfer agents, such as 4'-hydroxyacetanilide and other phenols asdescribed in U.S. Pat. No. 4,828,983 (issued May 9, 1989 to McClune).Electron transfer agents are compounds which facilitate the transfer ofone or more electrons between reactants in oxidation-reductionreactions. Many useful electron transfer agents are known in the art,such as phenazine methosulfate, phenazine ethosulfate, and benzo- andnaphthoquinones as described in U.S. Pat. No. 4,746,607 (issued May 24,1988 to Mura et al).

The components of the wash composition described above are readilyavailable commercially from a number of sources. Alternatively, they canbe prepared using known starting materials and procedures, as describedin U.S. Pat. No. 4,089,747 and other references noted above.

A preferred wash composition of this invention is buffered to a pH offrom about 6 to about 9 and comprises hydrogen peroxide, a phenolicelectron transfer agent, poly(vinylpyrrolidone), sec-butanol, andtriarylimidazole leuco dye chosen from the list of preferred leuco dyesshown above with2-(4-hydroxy-3,5-dimethoxyphenyl)-4,5-bis(4-methoxyphenyl)imidazolebeing most preferred.

A second composition of this invention is used for insolubilizing abiotinylated specific binding reagent or the complex formed fromreaction of such reactant with a specific binding partner. For example,this composition can be used to insolubilize the complex formed betweenan antigen and its corresponding biotinylated antibody. Alternatively, aternary complex of two antibodies (one being biotinylated) and anantigen can be insolubilized. Further still, a complex of biotinylatedantigen, or biotinylated anti-antibody with an antibody to be detectedcan be insolubilized.

The insolubilizing composition includes a particulate substrate to whichavidin is attached in a suitable chemical or mechanical means. Anyuseful substrate can be used as long as it is particulate,water-insoluble and does not adversely affect the assay. Suitableparticulate substrates are regular or irregular in shape and preparedfrom polymers, glass, ceramics, and other naturally occurring orsynthetically prepared materials. Polymeric particles, generally havinga diameter from about 0.1 to about 10 μm, are preferred. The substrateis suspended in an aqueous solution in an amount generally from about0.1 to about 5 percent solids. One or more buffers can be includedwithin the composition if desired.

Avidin is attached to the substrate using known technology includingcoating and drying, adsorption and chemical reaction. Generally, it iscovalently attached by reacting amino groups on the avidin molecule withactivated carboxy, activated haloalkyl, activated chloroethylsulfonyl orother reactive groups on the particles. A preferred procedure isdescribed in U.S.S.N. 315,086 (filed Feb. 24, 1989 by Sutton).

A reducing agent is also included in the insolubilizing composition sothat any unbound peroxidase will not prematurely oxidize the dye orleuco dye which is responsive to a peroxidative substance. Usefulreducing agents, include but are not limited to, ascorbic acid or saltsthereof (such as ascorbate), alkali bisulfites (such as sodiumbisulfite), water-soluble hydroquinones (such as hydroquinone sulfonicacid) and others readily apparent to one skilled in the art. Ascorbateis preferred. The reducing agent is generally present in an amount of atleast about 5 mmolar, and preferably from about 10 mmolar to about 200,mmolar.

The insolubilizing composition preferably includes one or morewater-soluble organic solvents having the same properties as thosedescribed above for the wash composition. The particular solvents usedin the two compositions can be the same or different, in the same ordifferent amounts. Preferably, the solvents used in a given assay arethe same.

Both the wash and insolubilizing compositions are prepared by mixing theindividual components together in any suitable manner and container.Each can be used immediately, or stored for later use, for example, in adiagnostic test kit.

A diagnostic test kit can include the wash composition described aboveas well as one or more other components, equipment, instructions and thelike needed for a specific binding assay. Particularly, the test kitincludes a receptor for the specific binding ligand. Other usefulcomponents of the kit include additional receptors (such as a secondreceptor which is biotinylated), labeled or unlabeled, labeled ligandanalogs, the immobilizing composition of this invention, materials whichbind specifically to the receptor or ligand, disposable test devices(described below), reagent containers, pipettes, prefilter devices, andother reagents known to one skilled in the art.

Disposable test devices generally comprise a water-insoluble substratehaving one or more test zones (such as test wells). The substrate isprepared from a water-insoluble material such as glass, polymericmaterials, cellulosic materials and other materials known in the art.The device can be a test tube, petri dish, filter paper or test striphaving the zones for reaction. It can also be a microtest plate having amultiplicity of preformed test wells. Particularly useful test devicesare described and claimed in U.S. Pat. No. 4,870,007 (issued Sep. 26,1989 to Smith-Lewis), incorporated herein by reference, which areavailable commercially in diagnostic test kits identified as Surecell™test kits by Eastman Kodak Co.

The present invention provides a method whereby a detectable complexbetween a ligand (a substance to be detected) and a receptor (a compoundwhich reacts specifically with the ligand) is obtained. Advantageously,the method is simple and therefore can be performed in a doctor's officeor in a consumer's home to provide immediate results. The test can beused to detect the presence or absence of a mono- or multivalent ormultideterminant ligand in an aqueous liquid, such as a biologicalfluid.

More specifically, the present invention can be used in thedetermination (qualitative or quantitative measurement) of a ligand inaqueous liquids to which there are naturally occurring or syntheticallyproduced specific binding receptors. This determination can be made bymerely determining the presence or absence of the ligand, or byquantitatively determining the amount of ligand. In particular, theinvention can be used to assay biological fluids of animals, humans orplants, but preferably of humans. Such fluids include, but are notlimited to, whole blood, plasma, sera, lymph, bile, urine, spinal fluid,seminal fluid, lacrimal fluid, vaginal secretions, sputum, perspirationand the like as well as stool specimens. It is also possible to assayfluid preparations of human or animal tissue such as skeletal muscle,heart, kidney, lungs, brains, bone marrow, skin and the like.

The ligand of interest can be an immunological species which is (1) anysubstance which, when presented to an immunocompetent host, will resultin the production of a specific antibody capable of binding with thatsubstance, or (2) the antibody so produced, which ligand participates inan antigen-antibody reaction.

Representative ligands detectable with the present invention includeprimary amines, amino acids, peptides, polypeptides, proteins,lipoproteins, glycoproteins, drugs, haptens, enzymes, steroids, lipids,nucleic acids, hormones, vitamins, polysaccharides, glycolipids,alkaloids, organisms (bacteria, protozoa, fungi, viruses includingretroviruses, rickettsia and the like) and components thereof, bloodcomponents, tissue and organ antigens and other materials known to oneskilled in the art. In some instances, the ligand is an antibody whichis directed against a drug, hormone, antibiotic or other compound havingantigenic properties. Alternatively, the ligand can be an antigenicmaterial. In still another embodiment, the immunological species is anantibody which is directed against another antibody (that is, ananti-antibody). Both monoclonal and polyclonal antibodies can be used,and they can be whole molecules or various fragments thereof.Preferably, monoclonal antibodies are used in the assays.

In a preferred embodiment, the method is useful for the detection of hCGin urine or blood as an early indicator of pregnancy. In thisembodiment, one or more different antibodies to hCG are immobilized inthe test device in order to provide reagents for forming a complex withhCG at different epitopic sites. This embodiment is described in moredetail in U.S. Pat. No. 4,870,007 (noted above).

Generally, the method of this invention is carried out by contacting anenzyme-labeled receptor for a ligand of interest with a sample of liquidsuspected of containing the ligand in such a manner as to form areaction product (that is, complex) of any ligand present and theenzyme-labeled receptor. Generally, the liquid sample is applied to atest zone of a test device or placed in a test well, depending upon theconfiguration of the device. The presence or absence of the reactionproduct is then determined in a suitable manner after washing thecomplex with the wash composition of this invention, separatingunreacted materials from the reaction complex. Dye is provided from thecomposition in the presence of the substrate and dye providing reagents.

The method of the invention can be a competitive binding immunoassayusing both enzyme-labeled and unlabeled receptor. Either bound (that is,complexed) or unbound (that is, uncomplexed) materials can bedetermined. Physical separation of bound and unbound materials, ifdesired, can be carried out using any suitable separation equipment andthe wash composition of this invention.

In another embodiment, a competitive immunoassay uses a receptor for theligand and a fixed quantity of enzyme-labeled ligand. Complex formed anddetected using the wash composition of this invention is inverselyproportional to the amount of ligand in the specimen.

In still another embodiment, the receptor is unlabeled, and theligand-receptor complex is detected using an enzyme-labeled specificbinding reagent which specifically binds to the receptor. For example,if the ligand is an antigenic material, and the receptor is an unlabeledantibody, the labeled specific binding reagent could be ananti-antibody.

In any of these embodiments, the detectable complex can be reacted witha specific binding reagent which complexes with either the ligand orreceptor therefor, and which is either insoluble or capable ofinsolubilizing the complex. For example, the reagent can be avidinattached to an insoluble substrate (particulate or not) if either theligand or receptor is biotinylated.

In a preferred embodiment, the method is what is known in the art as animmunometric assay. The details of such assays are provided in U.S. Pat.No. 4,486,530 (noted above). Such an assay can be used to determinemultivalent or multideterminant ligands as described above, that isligands having two or more epitopic sites for immunological reactionwith two or more, same or different, receptor molecules. In the sandwichassay, a second receptor is brought into contact with the ligand eitherprior to, simultaneously with or subsequent to contact of the ligandwith a first receptor. The result is the formation of a complex of thetwo receptors with the ligand at least one of which is enzyme-labeled.Preferably, a second receptor is biotinylated. The resulting complex isinsolubilized using the insolubilizing composition of this invention asthe biotinylated receptor and the avidin on the particulate substratereact, and the resulting insolubilized complex can be separated fromunreacted material in a suitable manner. The other receptor in theinsolubilized complex is detectable from the enzyme.

In a preferred embodiment, a method for the determination of hCG in anaqueous specimen (urine or blood) comprises the steps of:

A. contacting a specimen suspected of containing human chorionicgonadotropin with a first antibody (preferably, biotinylated antibody)to hCG to form an immunological complex,

B. simultaneously with or subsequently to step A, contacting thespecimen with a second antibody to hCG to form a sandwich complex, thefirst and second antibodies being reactive at different epitopes, and atleast one of the antibodies being labeled with peroxidase, and the otherantibody being either insolubilized or capable of becoming so,

C. separating uncomplexed materials from the sandwich complex through afiltration membrane,

D. contacting the separated sandwich complex with the buffered aqueouswash composition described herein while in the presence of hydrogenperoxide, and

E. detecting the resulting dye as an indication of the presence of hCGin the specimen.

This method can be practiced in a doctor's office or at home for earlydetermination of pregnancy by assaying urine samples.

The following examples are representative of the practice of thisinvention and is not intended to limit the scope of the invention. Allpercentages are by weight unless otherwise indicated. Materials:

PVP is poly(1-vinyl-2-pyrrolidone) (MW=40,000) which was obtained fromGAF Chemical Corp.

Human chorionic gonadotropin was obtained from Calbiochem.

A biotinylated antibody was prepared using monoclonal anti-hCGantibodies purchased from Immuno-Search, Inc. and biotinN-hydroxysuccinimide purchased from Calbiochem-Behring Corp. followingthe procedure described by Hofmann et al, J.A.C.S. 100, p. 3585 (1978).

The peroxidase-labeled antibody was prepared using monoclonal anti-hCGantibodies purchased from Cambridge Medical Diagnostics and horseradishperoxidase purchased from Miles, Inc. following the procedure describedby Yositake et al (Eur. J. Biochem., 101, p. 395, 1979).

Succinylated casein was prepared by reacting casein with an equal weightof succinic anhydride for four hours at 25° C., then purifying theproduct by dialysis.

Other materials used in the examples were obtained from Eastman KodakCo. or Sigma Chemical Co.

EXAMPLE 1: WASH COMPOSITION

The following composition was prepared and used as a wash in thedetermination of hCG as described in Example 3 below.

A solution of 2-4-(hydroxy-3,5-dimethoxyphenyl)-4,5-bis(4-methoxyphenyl)imidazole leuco dye (0.2% leuco dye) and polyvinyl pyrrolidone (20%)in water was prepared. A sample of this solution (5 ml) was added to 50ml of a solution comprising sodium dihydrogen phosphate (20 mmolar, pH7.2), diethylenetriaminepentaacetic acid chelating agent (20 μmolar),hydrogen peroxide (16 mmolar), 4'-hydroxyacetanilide electron transferagent (8 mmolar), sec-butanol (15 ml) and water (30 ml). This providesabout 15 volume percent of sec-butanol. The final concentration of leucodye was 0.01% and that of polyvinyl pyrrolidone was 1%.

EXAMPLE 2: INSOLUBILIZING COMPOSITION

An insolubilizing composition of this invention was prepared bysuspending particles of poly[styrene-co-m &p-(2-chloroethylsulfonylmethyl)styrene] (96:4 molar ratio) (0.6 percentsolids) in phosphate buffer (250 μl, 100 mmolar, pH 7.2). Avidin hadbeen covalently attached to the particles through the reactive2-chloroethyl groups on the particles. Also included in the compositionwere ascorbate (25 mmolar) and sec-butanol (2.5 volume percent).

EXAMPLE 3: ASSAY FOR HCG USING A WASH COMPOSITION TO PROVIDE A DYE

The wash composition of Example 1, and the insolubilizing composition ofExample 2 were used in an assay for hCG in the following manner.

A disposable test device like that described in U.S. Pat. No. 4,870,007(noted above) having a 5 μm commercially available nylon filter membranein each of three test wells was used in the assay. Each membrane hadbeen coated with succinylated casein. The biotinylated antibody (3 μg)to hCG immobilized within polyacrylamide binder (60 μg) was coated inone of the test wells. 3-(N-morpholine)propanesulfonic acid (pH 7.5)buffer was dried in a separate location in this test well. A second testwell containing dried buffer (2 mg) in polyacrylamide binder (60 μg) wasused as a negative control. The third well contained dried hCG (400mI.U.) in a separate location from a dried coating of biotinylatedanti-hCG antibodies (3 μg) in polyacrylamide binder (60 μg) and buffer(2 mg), as a positive control.

A urine specimen (about 200 μl), prefiltered to remove impurities, andcontaining about 50 mI.U./ml of hCG was added to each well of the testdevice, followed by addition of peroxidase-labeled anti-hCG antibodies(40 μl of a 10⁻⁹ molar solution). After a one minute incubation, theinsolubilizing composition of Example 2 (40 μl of a 0.6% dispersion) wasadded to each well and the fluid was allowed to drain through themembrane of each well.

Uncomplexed materials were washed through the membranes using the washcomposition of Example 1 (110 μl per well). After 2 minutes ofincubation, the dye formed on the membranes was measured visually toshow a positive test for hCG in the specimen.

EXAMPLE 4: ASSAY FOR HCG USING ASCORBATE AS THE REDUCING AGENT ANDACETONITRILE AS THE WATER-SOLUBLE SOLVENT

This example illustrates as assay for hCG using a wash compositioncontaining acetonitrile as the water-soluble solvent and ascorbate asthe reducing agent.

The disposable test device used was the same as that described inExample 3.

A wash composition was prepared from the following components: leuco dye(0.01% as in Example 1), polyvinyl pyrrolidone (1%), sodium dihydrogenphosphate (100 mmolar, pH 7.2), diethylenetriaminepentaacetic acid (10μmolar), 4'-hydroxyacetanilide (4 mmolar), hydrogen peroxide (8 mmolar)and acetonitrile (15 volume %).

The insolubilizing composition (prepared like that described in Example2) contained: avidin-particle composition (0.94% solids), sodiumascorbate (25 mmolar) glucose (18.5 mmolar), catalase (147 units/ml) andglucose oxidase (0.26 units/ml). The last three components keep theascorbate in reduced form as long as excess oxygen is prevented fromentering the solution.

The assay was carried out as described in Example 3 except that 28 μl ofthe insolubilizing composition and 160 μl of the wash composition wereadded to the test device wells.

The dye that formed on the membranes of the test device was evaluatedvisually as showing a positive test for hCG.

EXAMPLE 5: ASSAY FOR HCG USING ASCORBATE AS THE REDUCING AGENT ANDSEC-BUTANOL AS THE WATER-SOLUBLE SOLVENT

The assay described in Example 4 was repeated except that theinsolubilizing composition contained 200 mmolar ascorbate instead of 25mmolar. The dye that formed on the test device membranes was evaluatedvisually as showing a positive result for hCG.

EXAMPLE 6: ASSAY FOR hCG USING HYDROQUINONE SULFONATE AS THE REDUCINGAGENT AND SEC-BUTANOL AS THE WATER-SOLUBLE SOLVENT

This example illustrates an assay for hCG using a wash composition asdescribed in Example 4, except that the water-soluble organic solventused was sec-butanol (15 volume %). The insolubilizing composition,prepared as described in Example 2, contained an avidin-particlecomposition (0.94% solids) and hydroquinone sulfonate (10 μmolar) as thereducing agent.

The assay showed a positive indication of hCG on the test devicemembranes.

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be effected within the spirit and scope of theinvention.

I claim:
 1. A method for the determination of a specific binding ligandcomprising the steps of:A. contacting a specimen suspected of containingsaid specific binding ligand with an enzyme-labeled receptor for saidspecific binding ligand to form a detectable complex of ligand andreceptor, B. separating uncomplexed receptor from said detectablecomplex by washing said detectable complex with a buffered aqueous washcomposition comprising:a. a composition which provides a dye in responseto said enzyme, b. a buffer, and c. a water-soluble organic solventwhich has a molecular weight between about 40 and about 100 and ispresent in an amount of from about 2.5 to about 25 volume %, and C.detecting the resulting dye as an indication of the presence of saidspecific binding ligand in said specimen.
 2. The method of claim 1wherein said receptor is labeled with peroxidase, and said dye-providingcomposition comprises one or more reagents which react to provide a dyein the presence of peroxidase.
 3. The method of claim 1 wherein saiddetectable complex is reacted with a specific binding reagent whichbinds specifically with either said ligand or said receptor, and saidbinding reagent is insoluble or capable of insolubilizing said complex.4. The method of claim 1 wherein said dye is detected on a microporousmembrane through which uncomplexed materials are washed.
 5. The methodof claim 1 wherein said buffered aqueous wash composition is buffered toa pH of from about 6 to about 9, said dye-providing compositioncomprises a triarylimidazole leuco dye, and said water-soluble organicsolvent is selected from the group consisting of lower alcohols,acetonitrile, ketones, and ethers, said buffered aqueous washcomposition further comprising an electron transfer agent.
 6. A methodfor the detection of human chorionic gonadotropin comprising the stepsof:A. contacting a specimen suspected of containing human chorionicgonadotropin with a first antibody directed to hCG to form animmunological complex, B. simultaneously or subsequently to step A,contacting said specimen with a second antibody to hCG to form asandwich complex, said first and second antibodies specifically bindingto different epitopes, and at least one of said antibodies being labeledwith peroxidase, and the other antibody either insolubilized or capableof becoming so, C. separating uncomplexed materials from said sandwichcomplex through a filtration membrane by washing said sandwich complexwith a buffered wash composition while in the presence of hydrogenperoxide, said wash composition comprising:a. a composition whichprovides a dye in response to peroxidase, b. a buffer, and c. awater-soluble organic solvent which has a molecular weight between about40 and about 100 and is present in an amount of from about 2.5 to about25 volume %, and D. detecting the resulting dye on said membrane as anindication of the presence of hCG in said specimen.
 7. The method ofclaim 6 wherein said wash composition further comprises hydrogenperoxide and an electron transfer agent.
 8. The method of claim 6wherein said other antibody is biotinylated, and is insolubilized bycontact with an insolubilizing composition comprising a particulatesubstrate having avidin attached thereto, and a reducing agent.
 9. Themethod of claim 7 wherein said reducing agent is selected from the groupconsisting of ascorbic acid, a salt thereof, an alkali bisulfite and awater-soluble hydroquinone.
 10. The method of claim 7 wherein saidinsolubilizing composition further comprises a water-soluble organicsolvent which has a molecular weight between about 40 and about 100 andis present in an amount of from about 2.5 to about 25 volume %.